Method and apparatus for amplification of nucleic acid sequences by using thermal convection

ABSTRACT

The present invention provides a nucleic acid sequence amplification method and apparatuses thereof that are simple in the design and easy to miniaturize and integrate into complex apparatuses, with capability of using DNA polymerases that are not thermostable. In the present invention, a plurality of heat sources are combined to supply or remove heat from specific regions of the sample such that a specific spatial temperature distribution is maintained inside the sample by locating a relatively high temperature region lower in height than a relatively low temperature region.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 10/801,342, filed Mar. 15, 2004 (pending), which isa continuation-in-part application claiming benefit of priority toPCT/KR02/01728, filed on Sep. 14, 2002, the contents of which areincorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention generally relates to methods and apparatuses foramplifying nucleic acid sequences. More particularly, it relates tomethods and apparatuses using thermal convection, in which temperaturecontrolled amplification processes including the polymerase chainreaction (PCR) and related processes can be performed to amplify targetnucleic acid sequences from genetic samples containing DNA or RNA.

BACKGROUND ART

Nucleic acid sequence amplification technology has a wide application inbioscience, genetic engineering, and medical science for research anddevelopment and diagnostic purposes. In particular, the nucleic acidsequence amplification technology using PCR (hereafter referred to as“PCR amplification technology”) has been most widely utilized. Detailsof the PCR amplification technology have been disclosed in U.S. Pat.Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188.

Various apparatuses and methods incorporating automated PCRamplification processes have been developed and used for fast andefficient amplification of a variety of genetic samples. The basicworking principle of such technology is as follows.

In the commercialized PCR amplification technology, a sample is preparedto contain a template DNA to be amplified, a pair of oligonucleotideprimers complementary to a specific sequence of each single strand ofthe template DNA, a thermostable DNA polymerase, and deoxynucleotidetriphosphates (dNTP). A specific portion of the nucleic acid sequence ofthe template DNA is then amplified by repeating a temperature cycle thatsequentially changes the temperature of the sample. Typically, thetemperature cycle consists of three or two temperature steps, and theamplification processes during the temperature cycle occur in thefollowing manner.

The first step is the denaturation step in which the sample is heated toa high temperature and double stranded DNAs become separated to singlestranded DNAs. The second step is the annealing step in which the sampleis cooled to a low temperature and the single stranded DNAs formed inthe first step bind to the primers, forming partially double strandedDNA-primer complexes. The last step is the polymerization step in whichthe sample is maintained at a suitable temperature and the primers inthe DNA-primer complexes are extended by the action of the DNApolymerase, generating new single stranded DNAs that are complementaryto each of the template DNA strands. The target nucleic acid sequencesas selected by the sequences of the two primers are replicated duringeach cycle consisting of the above three steps. Typically, severalmillions or higher number of copies of the target nucleic acid sequencescan be produced by repeating the temperature cycles for about 20 to 40times.

The temperature of the denaturation step is typically 90-94° C. Thetemperature of the annealing step is controlled appropriately accordingto the melting temperatures (T_(m)) of the primers used, and ittypically ranges from 35 to 65° C. It is typical to set the temperatureof the polymerization step to 72° C. and use a three-step temperaturecycle, since the most frequently used Taq DNA polymerase (a thermostableDNA polymerase extracted from Thermus aquaticus) has the optimalactivity at that temperature. A two-step temperature cycle in which thepolymerization temperature is set to the same as the annealingtemperature, can also be used since the Taq DNA polymerase has a broadtemperature range of the polymerase activity.

In the most widely used method, a reaction vessel containing the sampleis made in contact with a solid metal block having a high thermalconductivity, and the temperature of the solid metal block is changed bycombining it with heating and cooling devices to achieve the desiredtemperature cycling of the sample. The commercial products adopting thistype of methods often use a gold-plated silver block that has very highthermal conductivity and/or the Peltier cooling method in order toachieve rapid temperature change. Recently, methods using a fluid suchas gas or liquid as a heat source instead of the solid metal block, havebeen developed to achieve rapid temperature change, and products usingsuch methods are being commercialized. In this type of methods, a fluidheated to a suitable temperature is circulated around the reactionvessel in a manner that an efficient thermal contact can be providedbetween the fluid heat source and the reaction vessel containing thesample. Other types of methods have also been developed to achieve rapidtemperature cycling. Additional examples include a method of contactingthe reaction vessel containing the sample or the sample itselfsequentially with multiple heat sources each at a specific temperature,a method of heating the sample directly with infrared radiation, etc.

The prior nucleic acid sequence amplification apparatuses have a numberof drawbacks as they operate to change the temperature of the wholesample according to the three- or two-step temperature cycle.

Firstly, the prior nucleic acid sequence amplification apparatuses ofthe temperature cycling type are complex in their design since processesfor changing the sample temperature are necessary. In order to performsuch temperature change processes, the method incorporating a solidmetal block or a fluid as a heat source requires a means for controllingand changing the temperature of the heat source rapidly and uniformlyand also a means for controlling the time interval of the temperaturechange. Similarly, the method of contacting the reaction vessel or thesample sequentially with multiple heat sources each at a specifictemperature requires a means for moving the reaction vessel or thesample quickly and precisely and also a means for controlling the movingtime and interval.

Secondly, it is difficult to integrate the prior nucleic acid sequenceamplification apparatuses in a complex apparatus or a miniaturizeddevice, due to their complicated design. Recently, miniaturized complexapparatuses are under development in the biotechnology field. Forexample, Lab-on-a-chip has been developed by integrating channels forsample passage, valves, pressure gauges, reaction vessels, detectionunits, etc. as a single unit on a glass, silicon, or polymer plate usingphotolithography. Such miniaturized complex apparatuses are expected tohave wide applications for various research and medical purposes. In thecase that a nucleic acid sequence amplification apparatus needs to beintegrated to such miniaturized chip, the prior method has a drawback inminiaturization because it requires a complex design to enable thetemperature change processes. Furthermore, it is difficult to integratethe prior apparatuses in a complex apparatus in which rapid temperaturechange is not desirable.

Thirdly, the prior nucleic acid sequence amplification apparatuses canonly use thermostable DNA polymerases such as Taq DNA polymerase. Thisis because the prior apparatuses have the process of heating the wholesample to a high temperature.

Finally, the prior nucleic acid sequence amplification apparatuses havea limitation for reducing the PCR reaction time. Since the priorapparatuses require the processes for changing the temperature of thewhole sample, the PCR reaction time must take more time at least as muchas the time needed for the temperature change.

SUMMARY OF THE INVENTION

The present invention is contrived to solve the above problems. It is anobjective of the present invention to provide a new nucleic acidsequence amplification method and apparatuses thereof based on thermalconvection. The new method and apparatuses according to the presentinvention achieve amplification of nucleic acid sequences by forming aplurality of specific regions having different temperatures inside thesample and thereby causing natural thermal convection of the sample tooccur as a result of the temperature gradient among the differentregions.

It is also an objective of the present invention to provide a method andapparatuses thereof that are simpler in their design and do not requirecomplex components such as a means for changing the temperature in acontrolled manner and a means for controlling the time interval of thetemperature change as are required in the prior temperature cyclingmethods and apparatuses.

Therefore, it is another objective of the present invention to provide anucleic acid sequence amplification method and apparatuses thereof thatare simpler than the prior art so that they can be readily miniaturizedand thus integrated into complex miniaturized apparatuses such asLap-on-a-chip.

It is still another objective of the present invention to provide anucleic acid sequence amplification method and apparatuses thereof basedon the thermal convection in which not only the thermostable DNApolymerases but also non-thermostable DNA polymerases can be used.

It is still further objective of the present invention to provide a moreefficient nucleic acid sequence amplification method and apparatusesthereof that do not require the temperature change processes needed inthe prior art.

Other objects and advantages of the invention will become clear to thoseskilled in the art from the following detailed description, claims, anddrawings.

In order to achieve the above objectives, the present invention providesa new nucleic acid sequence amplification method and apparatuses thereofbased on the novel thermal convection type operation principle describedbelow.

To achieve the above objectives, the present invention provides anucleic acid sequence amplification method using PCR, which methodcomprises:

a step of injecting into a reaction vessel a sample containing atemplate DNA having target nucleic acid sequences to be amplified, DNApolymerase, deoxyadenosine triphosphate, deoxycytidine triphosphate,deoxyguanosine triphosphate, deoxythymidine triphosphate, and at leasttwo oligonucleotide primers complementary to the 3′ terminus of each ofthe target nucleic acid sequences; and

a step of maintaining a specific spatial temperature distribution in thesample by contacting thermally with the sample a plurality of heatsources which supply heat to, or remove heat from specific regions ofthe sample such that a relatively high temperature region is locatedlower in height than a relatively low temperature region,

wherein the specific spatial temperature distribution comprises specificspatial regions each fulfilling a temperature condition suitable for (i)a denaturation step in which double stranded DNAs become separated tosingle stranded DNAs, (ii) an annealing step in which the singlestranded DNAs formed in the denaturation step hybridize to the primersto form DNA-primer complexes, or (iii) a polymerization step in whichthe primers in the DNA-primer complexes are extended by thepolymerization reaction,

and wherein the specific spatial temperature distribution is atemperature distribution that induces circulation of the sample bythermal convection so that the denaturation, annealing, andpolymerization steps occur sequentially and repeatedly inside thesample.

To achieve the above objectives, the present invention provides anucleic acid sequence amplification apparatus using PCR, which apparatuscomprises:

a plurality of heat sources which may supply heat to, or remove heatfrom a plurality of specific regions in a sample contained in a reactionvessel,

wherein the heat sources are arranged to maintain a specific spatialtemperature distribution in the sample such that a relatively hightemperature region is located lower in height than a relatively lowtemperature region,

wherein the specific spatial temperature distribution comprises specificspatial regions each fulfilling a temperature condition suitable for (i)a denaturation step in which double strand DNAs become separated tosingle strand DNAs, (ii) an annealing step in which the single strandDNAs formed in the denaturation step hybridize to the primers to formDNA-primer complexes, or (iii) a polymerization step in which theprimers in the DNA-primer complexes are extended by the polymerizationreaction,

and wherein the specific spatial temperature distribution is atemperature distribution that induces circulation of the sample bythermal convection so that the denaturation, annealing, andpolymerization steps occur sequentially and repeatedly inside thesample.

In the present invention, spatial regions are generated inside thereaction vessel containing the sample, in which regions thedenaturation, annealing, and polymerization steps can occur sequentiallyand repeatedly. In order to achieve this, a plurality of heat sourcesare combined to supply heat to, or remove heat from the specific regionsof the sample, and moreover a relatively high temperature region islocated to be lower in height than a relatively low temperature region.This results in generation of a natural thermal convection as a resultof the temperature gradient between the specific regions, therebycausing circulation of the sample among the different temperatureregions. Thus, the denaturation, annealing, and polymerization steps canoccur sequentially and repeatedly, resulting in amplification of nucleicacid sequences.

As described, the nucleic acid sequence amplification apparatuses of thepresent invention are based on the thermal convection method and it hasthe following characteristics in their design. Firstly, the apparatus ofthe present invention requires a plurality of heat sources that canmaintain a plurality of specific temperature regions in the sampleinside the reaction vessel at selected temperatures. Secondly, arelatively high temperature region should be positioned lower in heightthan a relatively low temperature region so as to induce circulation ofthe sample among the specific temperature regions via thermalconvection. More specifically, the sample in the high temperature regionhas a lower density than that in the low temperature region. Therefore,the buoyant force is generated and it causes the sample to move from thehigh temperature region at the lower position to the low temperatureregion at the higher position, while the gravitational force causes thesample to move in the opposite direction. A natural thermal convectionis thus generated by the temperature difference, resulting incirculation of the sample among the specific temperature regions.Finally, the temperatures of the specific temperature regions should beselected such that spatial regions, in which the denaturation,annealing, and polymerization steps can occur in each region, can beformed in the sample and also the three steps can be performedsequentially and repeatedly by thermal convection-induced circulation ofthe sample among the specific temperature regions at an appropriatespeed.

The objectives, features and advantages described above will be apparentfrom the following detailed description provided in connection with theattached drawings. In describing the present invention, detailedexplanation on the related prior art will be omitted when it canunnecessarily make the points of the present invention ambiguous.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic diagram of the operation principle of thenucleic acid sequence amplification method based on the thermalconvection.

FIGS. 2 a and 2 b show schematic diagrams of the cases having more thanthree specific temperature regions in the sample.

FIGS. 3 a and 3 b show a cross sectional view and a perspective view,respectively, of the nucleic acid sequence amplification apparatusaccording to the present invention.

FIG. 4 shows the temperature distribution of the sample at variousheights in the reaction vessel.

FIG. 5 is a photograph of the electrophoresis result illustratingresults of Example 1 at various reaction times.

FIG. 6 is a photograph of the electrophoresis result illustratingresults of Example 2 for each pair of primers.

FIG. 7 is a photograph of the electrophoresis result illustratingresults of Example 3 at various reaction times.

EXPLANATION ON THE NUMBERS OF THE IMPORTANT PARTS IN THE DRAWINGS

-   -   1, l′: High temperature region    -   2, 2′: Low temperature region    -   3, 4, 3′, 4′: Heat source    -   5: Convection region    -   6: Reaction vessel    -   101: First conduction block    -   102: Second conduction block    -   103: Reaction vessel    -   104: Heating device    -   105: Inlet of temperature control fluid    -   106: Outlet of temperature control fluid    -   107: Insulator    -   112, 117: Through hole    -   111: Opening

DETAILED DESCRIPTION OF THE INVENTION

As used herein, by “height” it is generally meant vertical height.

As used herein, “reaction vessel” refers to any container, which maycontain a sample comprising nucleic acid in which a PCR reaction mayoccur by thermal convection. The reaction vessel may be made of a widevariety of material so long as it is capable of conducting heat and isable to impart heat to or remove heat from the sample. The reactionvessel is not limited by size or shape so long as a PCR reaction iscapable of being carried out through thermal convection. For example,although FIG. 3 exemplifies what looks to be a straight cylindricalreaction vessel, the invention is not bound by any particular shape. Forinstance, the reaction vessel may be tapered from top to bottom or frombottom to top, so long as thermal convection is capable of beingestablished within the sample in the reaction vessel.

As used herein, the “first conduction block” refers to the heatconductive element that generally imparts heat to the sample.

As used herein, the “second conduction block” refers to the heatconductive element that generally removes heat from the sample. In thisregard, the apparatus depicted in FIG. 3 is for illustration only andvarious modifications and improvements are possible, so long as that thesecond conduction block is capable of removing heat from the sample atthe site of contact. In one embodiment, the apparatus depicted in FIG. 3may be modified to improve the thermal contact of the heat source withthe sample. For instance, the second thermally conductive block 102 thatworks as a cooling unit may be modified so as to make physical contactwith the sample itself instead of being in contact with the reactionvessel. In another embodiment, the shape of the second thermallyconductive block may be modified to comprise a plurality of protrusionsin the shape of dip sticks each of which may fit into the opening of thereaction vessel on the top and thus make physical contact with the upperportion of the sample.

In yet another embodiment, the second thermally conductive block may bemodified to comprise a plurality of receptors that fit to a plurality ofdip sticks. In this embodiment, each of the dip sticks may be installedin the opening on the top of the reaction vessel and make physicalcontact with the upper portion of the sample and also with one of thereceptors included in the second thermally conductive block.

BEST MODE FOR CARRYING OUT THE INVENTION

Below, the preferred embodiments according to the present invention areexplained in detail referring to the attached drawings.

FIG. 1 shows a schematic diagram of the operation principle of thenucleic acid amplification method based on the thermal convection. Theembodiment shown in FIG. 1 exemplifies the case in which a straighttubing with its one end closed is used as a reaction vessel and twospecific temperature regions 1 and 2 are generated. However, as shown inthe embodiments depicted in FIG. 2, reaction vessels having modifiedshapes may be used and three or more specific temperature regions may begenerated. It should be apparent to those skilled in the art thatvarious modifications including those described above may becontemplated based on the thermal-convection operation principle of thenucleic acid amplification method according to the present invention.

In one embodiment as shown in FIG. 1, the reaction vessel is in thermalcontact with two heat sources 3 and 4 that supplies heat to, or removesheat from the specific regions 1 and 2 in the sample directly orindirectly through the wall of the reaction vessel, thereby forming aspatial temperature distribution in the sample. The temperaturedistribution thus formed allows the three steps, the denaturation,annealing, and polymerization steps required in PCR to occur. Among thetwo regions 1 and 2 having different temperatures, the relatively hightemperature region 1 is positioned lower in height than the relativelylow temperature region 2. The temperature difference generates densitydifference in the sample. The buoyant force exerted on the low densitysample in the high temperature region 1 and the gravitational forceexerted on the high density sample in the low temperature region 2generate a thermal convection of the sample. Thus the sample naturallycirculates among the different spatial regions in each of which thedenaturation, annealing, and polymerization steps can occur. This designmakes the three PCR steps occur sequentially and repeatedly, therebyachieving amplification of DNA nucleic acid sequences by the PCRprocess. A more detailed operation is exemplified below.

For instance, the high temperature region 1 located at the bottom of thesample may be maintained at a temperature between 90 to 94° C. at whichtemperature double strand DNAs can be separated into single strand DNAs.Such arrangement makes the denaturation step occur mainly in the region1. The low temperature region 2 may be maintained at the annealingtemperature between 35 to 65° C. so that the DNAs denatured at the hightemperature region at the bottom portion moves to the low temperatureregion at the upper portion by thermal convection, and therefore thesingle stranded DNAs can anneal with the primers that are complementaryto the single stranded DNAs, forming DNA-primer complexes. In thisarrangement, if Taq DNA polymerase, known to have its optimal activityat 72° C. and a wide temperature range of activity even to lowtemperature, is used for polymerization, the polymerization step, whereDNA polymerase binds to the DNA-primer complex and the primer isextended, can occur in the low temperature region 2 and at the upperportion of the convection region 5. Therefore, the denaturation stepoccurs first in the high temperature region 1 and the denatured DNAsmove to the low temperature region 2 by thermal convection. Theannealing step thus occurs in the low temperature region in the presenceof the primers. The polymerization step finally occurs in the presenceof DNA polymerase during the time period that the DNA-primer complexesformed in the annealing step are passing through the low temperatureregion 2 and the convection region 5 by thermal convection.Consequently, the denaturation, annealing, and polymerization steps canoccur sequentially and repeated, thereby amplifying efficiently thetarget sequences of the sample DNA.

In other embodiments as shown in FIG. 2, it is contemplated that threespecific regions of the reaction vessel are in thermal contact with aplurality of heat sources. FIG. 2 a shows a schematic diagramillustrating one embodiment in which a plurality of heat sources 3, 3′,and 4 are arranged to form two high temperature regions 1 and 1′ and onelow temperature region 2. FIG. 2 b shows a schematic diagramillustrating another embodiment in which a plurality of heat sources 3,4, and 4′ are arranged to form one high temperature region 1 and two lowtemperature regions 2 and 2′. The plurality of the heat sources usedherein may be arranged separately for each temperature region or a sameheat source may be used for more than one temperature regions. In theembodiment illustrated in FIG. 2 a, if the two high temperature regions1 and 1′ are designed for the denaturation and polymerization steps,respectively, each region should be contacted with a heat source thatcan maintain the temperature of that region suitable for each step. Inthe embodiment illustrated in FIG. 2 b, if both of the two lowtemperature regions 2 and 2′ are designed for the annealing step, it ispreferable to use one heat source in replacement of the two heat sources4 and 4′. In addition, FIG. 2 b shows that it is possible according tothe present invention to construct a reaction vessel having separatesample inlet and outlet.

In order to improve the efficiency of the present invention, it isimportant to control the speed of the thermal convection such that thereaction at each step can occur sufficiently and at the same time thetotal reaction time can be reduced. This can be achieved by (a)controlling the temperature gradient between the specific temperatureregions, (b) controlling the diameter of the reaction vessel, or (c)changing the material of the reaction vessel. When controlling thetemperature gradient to adjust the thermal convection speed, it is mostconvenient to vary the temperature difference between the specifictemperature regions. However, this has a limitation since each of thespecific temperature regions has its own function for PCR that isdependent on temperature. Therefore, the distance between the hightemperature region (1 and 1′) and the low temperature region (2 and 2′)may be varied to obtain the same effect. For instance, the temperaturegradient becomes smaller as the distance between the two temperatureregions becomes larger if the temperature difference remains the same,and thus the thermal convection speed becomes reduced. Since theadhesion force between the wall of the reaction vessel and the sample isa factor that inhibits the thermal convection, the thermal convectionspeed can be controlled by adjusting the diameter of the reactionvessel. As the ratio of the surface area of the reaction vessel incontact with the sample relative to the volume of the sample becomeslarger, the adhesion force increases and the thermal convection speeddecreases. Therefore, the thermal convection speed can be controlled byadjusting the diameter of the reaction vessel, thereby controlling thesurface area of the reaction vessel in contact with the sample. Theadhesion force between the sample and the wall of the reaction vesselalso has an intimate relation with the material of the reaction vessel.Because the PCR process is normally performed in an aqueous solution,hydrophobic materials such as polyethylene and polypropylene that haveweaker adhesion force with water give rise to higher convection speedsas compared to hydrophilic materials such as glass. Therefore, theefficiency of the present invention can be improved further by designingthe reaction vessel suitable for the PCR reaction kinetics based on theprinciples described above.

FIG. 3 shows a cross sectional view (FIG. 3 a) and a perspective view(FIG. 3 b) of the nucleic acid sequence amplification apparatusaccording to one embodiment of the present invention. The apparatusshown in FIG. 3 comprises a plurality of heat sources as means formaintaining temperature, which include a heating unit; a cooling unit;or a combination of a heating unit and a cooling unit. Preferably, aninsulating means may be included in between the heat sources tothermally insulate the heat sources. In this particular embodiment, theapparatus comprises first and second heat sources that are in thermalcontact with specific regions of the sample. The first heat sourceconsists of a first thermally conductive block 101 and an electricheating unit 104 that supplies heat to the first thermally conductiveblock. The first thermally conductive block is in thermal contact withthe bottom of the reaction vessel to form a high temperature region atthe bottom of the sample. The second heat source consists of a secondthermally conductive block 102 and a circulating water bath thatcirculates water at certain temperature through the inside of the secondthermally conductive block to maintain the temperature of the secondthermally conductive block at a suitable temperature. The secondthermally conductive block 102 is in thermal contact with the upperportion of the reaction vessel to form a low temperature region at theupper portion of the sample. The second thermally conductive block 102comprises an inlet 105 though which water flows in from the water bath,an outlet 106 through which the water flows out, and a fluid circulationchannel for circulating the water inside the second thermally conductiveblock. Although the fluid circulation channel in the second thermallyconductive block is not depicted in FIG. 3, the person skilled in theart can understand that the fluid circulation channel is designed totransfer heat uniformly to the second thermally conductive block 102.The material of the thermally conductive blocks 101 and 102 is selectedto be copper that has a high thermal conductivity, and an insulator 107is inserted between the two blocks to prohibit direct heat transfer. Thefirst and second thermally conductive blocks 101 and 102 have receptoropenings for introduction of the reaction vessels. The receptor openingconsists of an opening 111 having its one end closed in the firstthermally conductive block 101, a through hole 112 in the secondthermally conductive block, and another through hole 117 in theinsulator.

In Example 1, 2, and 3 described later, the high temperature region atthe bottom of the sample is maintained at 94° C. by controlling theelectric heating unit 104, and the low temperature region at the upperregion of the sample at 45° C. by controlling the temperature of waterin the circulating water bath.

The present invention is not limited to the nucleic acid sequenceamplification apparatus depicted in FIG. 3. The following modificationsare possible.

Firstly, the structures of the thermally conductive blocks 101 and 102may be modified. For instance, the first thermally conductive block 101may be contacted thermally with the bottom portion of the reactionvessel and the second thermally conductive block 102 with the upperportion of the reaction vessel, while the middle of the reaction vesselmay be contacted with air or a third thermally conductive block. Inaddition, different from the embodiment depicted in FIG. 3 in which heatis transferred from the blocks to the specific regions of the samplethrough the wall of the reaction vessel, the thermally conductive blocksmay be contacted directly with the sample.

Secondly, the material of the thermally conductive blocks may bemodified. In the embodiment depicted in FIG. 3, the thermally conductiveblocks 101 and 102 made of copper are used, but the material is notlimited to copper. Nearly any material that can transfer heat to thereaction vessel may be used. For instance, other thermally conductivesolid or fluid such as liquid or gas may be used in replacement of thethermally conductive blocks used above. For some instance, infraredradiation or other means may be used in replacement of some or all ofthe thermally conductive blocks 101 and 102.

Thirdly, means for maintaining the temperatures of the first and secondthermally conductive blocks are not limited to a circulating water bathor an electric heating unit. Nearly any unit that can supply heat to,remove heat from the sample may be used.

Fourthly, nearly any means such as solid, liquid, or gas may be used inreplacement of the insulator 107 depicted in FIG. 3 as far as it issuitable for insulating heat transfer between conductive materials. Itis also possible to use a composition that does not include theinsulator.

Finally, when a modified reaction vessel (for example, those shown inFIG. 2 a or 2 b) is used instead of the reaction vessel illustrated inFIG. 1 to facilitate the thermal convection, one may use a plurality ofheat sources including thermally conductive blocks and theirmodifications that are suitably modified based on the principle of thepresent invention

The first, second, and third cases described above are examples in whicha part of the heat source, particularly the thermally conductive block,is modified. As used herein, the heat source refers to any means thatcan be used for maintaining the temperature of the sample at a specificvalue. Therefore, in addition to the modification examples of the heatsources described above, any device may be used as a heat source in thepresent invention as far as it can be used to maintain a specific regionof the sample at a selected temperature. The present invention includesnearly any apparatus that has a function of maintaining specific regionsof the sample at selected temperatures. This is because the presentinvention is characterized not by a particular design of the heatsources but by the special arrangement of the heat sources intended forgenerating a specific temperature distribution inside the sample thatenables the PCR process to occur sequentially and repeatedly.

More detailed designs of the modification examples described above maybe varied depending on the development of industrial technologies.Therefore, detailed explanations are omitted.

FIG. 4 shows a temperature distribution measured at various heights fromthe bottom of the reaction vessel, demonstrating the principle of thePCR process based on the thermal convection. The thermal convection is aphenomenon by which movement of fluid is induced by a density differencegenerated by difference in temperature. This type of convection isreferred to as a natural convection, distinguished from a forcedconvection where fluid is forced to move by a pump or a propeller. Theterm convection as used in the present invention always refers to anatural convection. For a natural convection to occur in the reactionvessel, the bottom portion of the sample in the reaction vessel shouldbe higher in temperature than the upper portion.

As can be seen in FIG. 4, when the first thermally conductive block 101contacting with the bottom portion of the reaction vessel is maintainedat 96° C. and the second thermally conductive block 102 contacting withthe upper portion at 45° C., the high temperature region (the regionwith the temperature higher than or equal to 90° C. in FIG. 4), the lowtemperature region (the region with the temperature near 50° C.)., andthe convection region (the region having a temperature gradient) areformed. The sample is subject to the denaturation step in the hightemperature region. The denatured sample then moves to the lowtemperature region across the convection region, in which the sample issubject to the annealing step. While staying in the low temperatureregion and moving back through the convection region from the lowtemperature region, the sample is subject to the polymerization step.Thermal convection causes the sample to circulate the three regionssequentially and repeatedly, thereby leading to amplification of nucleicacid sequences by PCR.

FIG. 7 shows the results obtained by using DNA polymerase immobilized onthe solid surface. The term “immobilized DNA polymerase” as used hereinis meant a DNA polymerase that is immobilized on a solid support withits polymerase activity preserved. Various methods may be used toprepare the immobilized DNA polymerase, but it should provide animmobilized DNA polymerase that has a high enough polymerase activity soas to enable detection of nucleic acid sequences amplified by PCR oftemplate DNAs. The immobilized DNA polymerase used in the examples ofthe present invention was prepared to preserve a high polymeraseactivity by using a method in which the active site of the DNApolymerase was masked by a DNA substrate and immobilized on a Au surfaceby covalent bonding. Detailed procedure of the immobilization method isdescribed in the example. The polymerase activity of the immobilizedenzyme as prepared by this method was high enough (about 60-80% comparedto the solution phase DNA polymerase) to use for PCR. However, theimmobilized DNA polymerase that can be used with the present inventionis not limited to those prepared by the method used in the example ofthe present invention, but includes those prepared by other methods.

In the nucleic acid sequence amplification method of the thermalconvection type according to the present invention, DNA polymerases thatare not thermostable, such as Klenow fragment and T7 DNA polymerase, maybe used in addition to the thermostable polymerases such as Taq DNApolymerase. This is due to the following fact. By the virtue of thecharacteristics of the present invention, the temperature of the totalsample does not change from a high temperature to a low temperature orvice versa repeatedly, but the specific regions in the sample aremaintained at constant temperatures. For instance, the upper portion ofthe sample may be maintained at a low temperature, whereas the bottomportion of the sample may be maintained at a high temperature. It ispossible to use DNA polymerase that is not thermostable, by locating theimmobilized DNA polymerase in the low temperature region or in the upperportion of the convection region near the low temperature region.

EXAMPLES

Example 1, 2, and 3 described below confirm that the objectives of thepresent invention can be achieved using a nucleic acid sequenceamplification apparatus of the present invention.

Example 1 1. Methods

1.1. Reaction Vessel

A glass tubing with its one end closed was used as a reaction vessel.The glass tubing had a length of 55˜60 mm, an inner diameter of 2 mm, anouter diameter of 8 mm, and a thickness of 3 mm at the bottom-sideclosed end. The inner wall of the glass tubing was coated withpolytetrafluoroethylene using a spray type coating material andthermally hardened.

1.2. Sample

pBluescript II KS(+) was used as a template DNA. The sample used in PCRcontained 40 ng of the template DNA, 40 pmol each of T3 primer(5′-ATTAACCCTCACTAAAG-3′) (SEQ ID NO: 1) and T7 primer(5′-AATACGACTCACTATAG-3′) (SEQ ID NO: 2), 4 nmol of dNTP, 1 pmol (5 U)of Taq DNA polymerase, and 250 nmol of MgCl₂ in 100 μl of 10 mM Trisbuffer at pH 8.3 containing 50 mM KCl.

1.3. Reaction Temperature and Reaction Time

Firstly, the first thermally conductive block 101 was heated with anelectric heating unit and maintained at 96° C., and the second thermallyconductive block 102 was maintained at 45° C. using a circulating waterbath. The sample prepared above was injected to the reaction vessel, andthe reaction vessel was then inserted into the receptor 111, 117, and112. The sample was allowed to react for a suitable time. During thereaction, the reaction vessel was pressurized to about 1.2 atm by addingnitrogen gas to prevent boiling of the sample solution.

1.4. Measurement of the Temperature Distribution in the Sample

The temperature in each region of the sample was measured under theabove reaction conditions. The tip of a thermocouple thermometer wasplaced every 2.5 mm from the bottom of the reaction vessel, and thetemperature was measured and recorded after sufficient time. An exampleof the temperature distribution of the sample in the reaction vessel isshown in FIG. 4.

2. Results

First, the measured temperature in each region of the sample in thereaction vessel under the above reaction conditions confirmed (see FIG.4) that a high temperature region above 90° C. for denaturation, a lowtemperature region around 50° C. for annealing, and a convection regionhaving a temperature gradient for induction of the thermal convectionare formed. Polymerization is expected to occur in the low temperatureregion and the upper portion of the convection region.

After the sample was incubated for a given reaction time under the abovereaction conditions, the reaction vessel was taken out and cooled. Thereaction products were analyzed by electrophoresis using 1.0% agarosegel. FIG. 5 is a photograph of the electrophoresis results obtained atthe reaction times up to 4 hours for every 30 min time interval. Thereaction product is a 164 by double stranded DNA. As can be seen in FIG.5, the PCR reaction reaches saturation before 90 min.

Example 2 1. Methods

In addition to T3/T7 primer pair, KS/U, KS/Pvu ∥, and KS/Nae | primerpairs were also examined in the experiments. The reaction time was setto 150 min, and other reaction conditions were the same as in Example 1.The sequences of the T3 and T7 primers were described in Example 1, andthe sequences of other primers are given as follows:

KS primer: 5′-CGAGGTCGACGGTATCG-3′ (SEQ ID NO: 3) U primer:5′-GTAAAACGACGGCCAGT-3′ (SEQ ID NO: 4) Pvu II primer:5′-TGGCGAAAGGGGGATGT-3′ (SEQ ID NO: 5) Nae I primer:5′-GGCGAACGTGGCGAGAA-3′ (SEQ ID NO: 6)

2. Results

As in Example 1, the reaction products were analyzed by electrophoresis.FIG. 6 is a photograph of the electrophoresis results of Example 2,where lanes 1, 2, 3, and 4 are the results obtained with T3/T7, KS/U,KS/Pvu ∥, and KS/Nae | primer pairs, respectively. It can be seen thatthe four primer pairs produced double stranded DNAs with correct sizesof 164 bp, 144 bp, 213 bp, and 413 bp, respectively.

Example 3 1. Methods

Instead of adding Taq DNA polymerase to the sample, Taq DNA polymerasewas immobilized on the surface of a Au wire and it was located in thelow temperature region. Other experimental conditions were the same asin Example 1.

The method used to immobilize the DNA polymerase is described below.

The 65 base single stranded DNA and the KS primer shown below were mixedin a pH 8.3 phosphate buffer at 1:1 molar ratio. The resulting solutionwas incubated at 94° C. for 10 min and then cooled down slowly below 35°C. During this process, the 65 base single stranded DNA and the KSprimer were annealed to form a partially double stranded DNA. Anappropriate number of moles of Taq DNA polymerase (AmpliTaq Gold)purchased from Perkin Elmer (U.S.A.) was then added to this solution andthe resulting mixture was incubated in a dry bath at 72° C. for 10 min.Then, the mixture was moved to a dry bath at 50° C. and incubated for 20min to finish preparation of a masked DNA polymerase in which thepartially double stranded DNA is bound to the active site of the DNApolymerase.

KS primer: (SEQ ID NO: 1) 5′-CGAGGTCGACGGTATCG-3′ 65-mer: (SEQ ID NO: 7)3′-CCAGCTGCCATAGCTATTTTCTTTTCTTTCTTAAGTTCTTTT CTTTTCCTAGGTGATCAAGATCT-5′

In order to have a maximum amount of immobilized DNA polymerase be 0.26pmol, Au wire having an outer diameter of 0.1 mm and a length of 4.7 cmwas prepared and used after manipulating it to a coil shape having anouter diameter of 1.5 mm and a length of about 4 mm. In order to ensurethe cleanness of the surface of the Au wire, it was washed with Piranhasolution for 10 ˜15 minutes at 60 ˜70° C. and was rinsed with deionizedwater and subsequently with absolute ethanol, right before using.

In order to introduce reaction groups for immobilization on the Ausurface, a monolayer of thiol molecules was formed on the Au surface byusing the Au—S bond formation reaction, that is, by using the thiolateformation reaction between a linker molecule having a thiol group andAu, to prepare a supporting material. In this reaction, a mixed solutioncontaining two kinds of thiol molecules having an immobilizationreaction group and a non-reactive group, respectively, was used. Themole fraction of the thiol molecule having the immobilization reactiongroup with respect to the total moles of the two thiol molecules wasselected to be 5%. In order to introduce a carboxyl immobilizationreaction group, 12-mercaptododecanoic acid having a relatively longalkyl chain was used as a linker molecule. As a thiol molecule having anon-reactive group, 6-mercapto-1-hexanol or 1-heptanethiol was used as amatrix molecule. The carboxyl immobilization reaction group wasintroduced on the surface of the Au wire by placing it in 100 μl of a 2mM mixed thiol solution in ethanol for 2 hours at room temperature andwashing it with absolute ethanol.

The Au wire on which the carboxyl immobilization reaction groups wereintroduced was placed in 120 μl of an ethanol solution containing 10 mMof 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mM ofN-hydroxysuccinimide (NHS) for 2 hours at room temperature. The carboxylgroup was activated by reacting with NHS in the presence of EDC and thusforming NHS-ester.

After activating the carboxyl groups of the thiol monolayer, the Au wirewas moved to the enzyme solution containing the active-site masked DNApolymerase. In this step, the activated carboxyl (NHS-ester) of thethiol monolayer reacted with the primary amine of the protein, formingan amide bond (—CO—NH—). As a result, the Taq DNA polymerase wasimmobilized on the supporting material.

2. Results

As in Example 1, the reaction products were analyzed by electrophoresis.FIG. 7 is a photograph of the electrophoresis results obtained at thereaction times up to 4 hours for every 30 min time interval. As can beseen in FIG. 7, the PCR reaction reaches saturation before 150 minutes.

From the results of Example 1, 2, and 3, the following points can beseen.

Firstly, the nucleic acid sequence amplification apparatus based on thethermal convection according to the present invention works efficiently.

Secondly, it was confirmed that the PCR process can be performed bylocating the DNA polymerase immobilized on a solid surface in the lowtemperature region or in the upper portion of the convection region byusing the nucleic acid sequence amplification apparatus based on thethermal convection according to the present invention. It was thusconfirmed that DNA polymerases that are not stable at high temperaturecan also be used.

It should be apparent to those skilled in the art that the presentinvention described above is not limited to the above embodiments andthe attached drawings and that various substitutions, changes, andmodifications are possible without departing from the technical ideas ofthe present invention. Therefore, the above embodiments andmodifications are only for illustration, and should not be interpretedto be limiting the present invention. The real scope of the presentinvention should be determined by the following claims and is notrestricted in any way by the specification.

As described above, in the present invention, a plurality of specificregions of the sample are maintained at specific temperatures, andthermal convection among the specific regions makes the sample circulateinside the reaction vessel. Thus, the denaturation, annealing, andpolymerization steps can be performed sequentially and repeatedly.Therefore, the following effects can be noted.

Firstly, the nucleic acid sequence amplification apparatus can bedesigned with a simple composition. The present invention does notrequire the process for changing the temperature of the sample.Therefore, the design according to the present invention can be madesimpler because complex devices included in the prior apparatuses forchanging and controlling the sample temperature are not required.

Secondly, the apparatus according to the present invention can bereadily miniaturized or integrated into a complex apparatus such asLab-on-a-chip to perform the PCR nucleic acid sequence amplificationprocess. It can also be incorporated into the apparatuses in whichtemperature change is not desirable.

Thirdly, DNA polymerases that are not thermostable can also be used.This is because immobilized DNA polymerases can be used in the presentinvention by locating them in a specific region inside the reactionvessel which region is maintained at a temperature suitable for thepolymerase activity. According to the present invention, when animmobilized DNA polymerase is used, PCR can be performed with theimmobilized DNA polymerase maintained at the temperature where thepolymerase is active. Therefore, according to the present invention,enzymes having their optimal activities at low temperature, such asKlenow fragment or T7 DNA polymerase, may also be used for the PCRprocess.

Finally, the reaction time for PCR can be reduced. In the presentinvention, there is no need to change the temperature of the totalsample. Thus the time needed for changing and controlling thetemperature of the whole sample can be saved.

1. A nucleic acid sequence amplification method using polymerase chainreaction (PCR), which method comprises: a step of injecting into areaction vessel a sample containing a template DNA having target nucleicacid sequences to be amplified, DNA polymerase, deoxyadenosinetriphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate,deoxythymidine triphosphate, and at least two oligonucleotide primerscomplementary to the 3′ terminus of each of the target nucleic acidsequences; and a step of maintaining a specific spatial temperaturedistribution in the sample by contacting thermally with the sample aplurality of heat sources which supply heat to, or remove heat fromspecific regions of the sample such that a relatively high temperatureregion is located lower in height than a relatively low temperatureregion, wherein the specific spatial temperature distribution comprisesspecific spatial regions each fulfilling a temperature conditionsuitable for (i) a denaturation step in which double stranded DNAsbecome separated to single stranded DNAs, (ii) an annealing step inwhich the single stranded DNAs formed in the denaturation step hybridizeto the primers to form DNA-primer complexes, or (iii) a polymerizationstep in which the primers in the DNA-primer complexes are extended bythe polymerization reaction, and wherein the specific spatialtemperature distribution is a temperature distribution that inducescirculation of the sample by thermal convection so that thedenaturation, annealing, and polymerization steps occur sequentially andrepeatedly inside the sample.
 2. The nucleic acid sequence amplificationmethod of claim 1, wherein at least one of the heat sources comprises athermally conductive solid in thermal contact with a specific region ofthe reaction vessel or the sample; and a heating unit that supplies heatto the thermally conductive solid, a cooling unit that removes heat fromthe thermally conductive solid, or a combination of the heating unit andthe cooling unit.
 3. The nucleic acid sequence amplification method ofclaim 1, wherein at least one of the heat sources comprises a liquid inthermal contact with a specific region of the reaction vessel; areceptor in which the liquid is to be contained; and a heating unit thatsupplies heat to the liquid, a cooling unit that removes heat from theliquid, or a combination of the heating unit and the cooling unit. 4.The nucleic acid sequence amplification method of claim 3, wherein atleast one of the heat sources further comprises a circulation unit thatcirculates the liquid around the reaction vessel.
 5. The nucleic acidsequence amplification method of claim 1, wherein at least one of theheat sources comprises a gas in thermal contact with a specific regionof the reaction vessel; a heating unit that supplies heat to the gas, acooling unit that removes heat from the gas, or a combination of theheating unit and the cooling unit; and a circulation unit thatcirculates the gas around the reaction vessel.
 6. The nucleic acidsequence amplification method of claim 1, wherein at least one of theheat sources is an infrared radiation generating unit that supplies heatdirectly to the sample.
 7. The nucleic acid sequence amplificationmethod of claim 1, which method uses a means for insulating heattransfer between the heating sources.
 8. A nucleic acid sequenceamplification apparatus using PCR, which apparatus comprises: aplurality of heat sources which may supply heat to, or remove heat froma plurality of specific regions in a sample contained in a reactionvessel, wherein the heat sources are arranged to maintain a specificspatial temperature distribution in the sample such that a relativelyhigh temperature region is located lower in height than a relatively lowtemperature region, wherein the specific spatial temperaturedistribution comprises specific spatial regions each fulfilling atemperature condition suitable for (i) a denaturation step in whichdouble strand DNAs become separated to single strand DNAs, (ii) anannealing step in which the single strand DNAs formed in thedenaturation step hybridize to the primers to form DNA-primer complexes,or (iii) a polymerization step in which the primers in the DNA-primercomplexes are extended by the polymerization reaction, and wherein thespecific spatial temperature distribution is a temperature distributionthat induces circulation of the sample by thermal convection so that thedenaturation, annealing, and polymerization steps occur sequentially andrepeatedly inside the sample.
 9. The nucleic acid sequence amplificationapparatus of claim 8, wherein at least one of the heat sources comprisesa thermally conductive solid in thermal contact with a specific regionof the reaction vessel or the sample; and a heating unit that suppliesheat to the thermally conductive solid, a cooling unit that removes heatfrom the thermally conductive solid, or a combination of the heatingunit and the cooling unit.
 10. The nucleic acid sequence amplificationapparatus of claim 8, wherein at least one of the heat source comprisesa liquid in thermal contact with a specific region of the reactionvessel; a receptor in which the liquid is to be contained; and a heatingunit that supplies heat to the liquid, a cooling unit that removes heatfrom the liquid, or a combination of the heating unit and the coolingunit.
 11. The nucleic acid sequence amplification apparatus of claim 10,wherein at least one of the heat sources further comprises a circulationunit that circulates the liquid around the reaction vessel.
 12. Thenucleic acid sequence amplification apparatus of claim 8, wherein atleast one of the heat sources comprises a gas in thermal contact with aspecific region of the reaction vessel; a heating unit that suppliesheat to the gas, a cooling unit that removes heat from the gas, or acombination of the heating unit and the cooling unit; and a circulationunit that circulates the gas around the reaction vessel.
 13. The nucleicacid sequence amplification apparatus of claim 8, wherein at least oneof the heat sources is an infrared radiation generating unit thatsupplies heat directly to the sample.
 14. The nucleic acid sequenceamplification apparatus of claim 8, which method uses a means forinsulating heat transfer between the heating sources.
 15. The methodaccording to claim 1, wherein the heat source is shaped to comprise atleast one protrusion that fits in an opening of the reaction vessel,wherein said protrusion contacts the sample.
 16. The apparatus accordingto claim 8, wherein the heat source is shaped to comprise at least oneprotrusion that fits in an opening of the reaction vessel, wherein saidprotrusion contacts the sample.